Effect of Chitosan Addition in Whey-based Biodegradable Films

Cristiane Leonardelli Wendel Paulo Silvestre Camila Baldasso About the authors

Abstract

Whey, a by-product of dairy industry, is a feedstock widely employed in the production of biodegradable films. However, these films present some limitations when considering the performance of synthetic polymers, especially biological transformation by decomposition. This work aimed to evaluate the effects of chitosan addition to whey-based films to improve films physical-chemical properties and resistance to microbial degradation. The results showed that there was an interaction effect between the chitosan concentration and the storage time for the physical-chemical properties of elongation at break and opacity. There was statistical difference among the formulations; however, for the moisture content and film thickness, there was no interaction effect between the formulation and the storage time. The films with 1.5 and 3.0 wt.% chitosan presented a yellowish hue, characteristic of the polysaccharide; this could also be detected by SEM analysis. The films presented an excellent biodegradability, being decomposed in about 8 days. Considering all chitosan contents tested had similar performances, the chitosan content of 0.15 wt.% was the one with the better cost-benefit relation.

Keywords:
biopolymers; biodegradable films; antimicrobial agent; shelf life

INTRODUCTION

Cheese is one the most produced and consumed dairy products in the world. In its production, there is also the production of whey, which can also be obtained by casein extraction [11 Florentino ER, Macedo GR, Santos ES. Caracterização do soro de queijo visando processo de aproveitamento. Hig. Aliment. 2005;19:30-2.]. It is estimated that, on average, for each kilogram of produced cheese, are used 10 L of milk, being obtained 9 L of whey [22 Neves BS. Aproveitamento de subprodutos da indústria de laticínios. In: EMBRAPA. Sustentabilidade da pecuária de leite no Brasil: qualidade e segurança alimentar. Brasília: EMBRAPA; 2001. p. 97-108.].

Whey represents about 90% of the milk amounts used in dairy products and casein fabrication, having a low value and a high biochemical oxygen demand (BOD); this renders whey a product whose treatment and disposal is somewhat complicated. Aiming to minimize these impacts and also due to the considerable amounts of nutritional compounds, whey is being used in the research and development of new products, such as in food industry [33 Zavareze ER, Moraes KS, Salasmellado MM. Qualidade tecnológica e sensorial de bolos elaborados com soro de leite. Ciênc. Tecnol. Aliment. 2010; 30:102-6.], ethanol production [44 Florêncio IM, Florentino ER, Silva FLH, Martins RS, Cavalcanti MT, Gomes JP. Produção de etanol a partir de lactossoro industrial. Rev. Bras. Eng. Agric. Amb. 2013; 17:1088-92.], production of polymeric edible films [55 Dianin, IMB. Aplicação de proteínas do soro de leite na formulação de biofilme comestível para aplicação em frutas [dissertation]. Londrina: UNOPAR; 2016.], among other uses.

Although the concentrated whey has market as a supplement and protein source, the process of water removal is quite expensive, which may render the concentrated product (dry whey) economically unsuitable for film and packaging applications [66 Balagtas JV, Hutchinson FM, Krochta JM, Summer DA. Anticipating Market Effects of New Uses for Whey and Evaluating Returns to Research and Development. J. Dairy Sci. 2003; 86:1662-72.,77 Keogh C, Li C, Gao Z. Evolving consumer trends for whey protein sports supplements: the Heckman ordered probit estimation. Agric. Food Econ. 2019; 7(6):1-10.]. A possible way to save production costs is to use the whey in the diluted form (or only partially dried to increase whey concentration), generally a byproduct from waste streams of dairy and food industries. This approach may render a waste that needs special treatment prior to disposal to a feedstock for the production of films, membranes, and packaging [66 Balagtas JV, Hutchinson FM, Krochta JM, Summer DA. Anticipating Market Effects of New Uses for Whey and Evaluating Returns to Research and Development. J. Dairy Sci. 2003; 86:1662-72.,88 Ramos OL, Fernandes JC, Silva SI, Pintado ME, Malcata X. Edible Films and Coatings from Whey Proteins: A Review on Formulation, and on Mechanical and Bioactive Properties. Crit. Rev. Food Sci. Nutr. 2012; 52:533-52.].

Whey-based biopolymers can form films by changing the solution physical-chemical conditions, such as salt addition, heating, and pH-changing. The main method for film production is by solubilizing the biopolymer, followed by solvent evaporation. Solvent evaporation allows the polymeric chains to interact by electrostatic interaction, hydrogen bonding, and Van der Waals forces, generating the polymeric structure [99 Onwulata CI, Huth PJ. Whey Processing, Functionality and Health Benefits. Ames: Blackwell Publishing and The Institute of Food Technologists; 2008.].

Due to some properties, whey-based films were studied as active and biodegradable packaging for food products [99 Onwulata CI, Huth PJ. Whey Processing, Functionality and Health Benefits. Ames: Blackwell Publishing and The Institute of Food Technologists; 2008.]. However, these films may suffer microbial degradation during the product shelf life. To avoid this, several natural antimicrobial agents were proposed and tested; among them can be cited garlic and oregano essential oils and extracts [1010 Seydim AC, Sarikus G. Antimicrobial activity of whey protein based edible films incorporated with oregano, rosemary and garlic essential oils. Food Res Int. 2006; 39:639-44.], chitosan [1111 Barros-Velazquez J. Antimicrobial Food Packaging. San Diego: Academic Press; 2016.-1212 Aider M. Chitosan application for active bio-based films production and potential in the food industry: Review. LWT - Food Sci. Technol. 2010; 43:837-42.], nisin [1313 Pintado CMBS, Ferreira MASS, Sousa I. Properties of Whey Protein-Based Films Containing Organic Acids and Nisin To Control Listeria monocytogenes. J. Food Prot. 2009; 72:1891-6.], and organic acids [1414 Manab A, Sawitri ME, Al-Awwaly KU, Purnomo H. Antimicrobial activity of whey protein based edible film incorporated with organic acids. African J. Food Sci. 2011; 5:6-11.]. Among these, chitosan highlights itself to be used in whey-based films.

Chitosan is a biopolymer, obtained by the alkaline deacetylation of chitin, which constitute the exoskeleton of crustaceans and arachnids. Its biodegradable, antimicrobial, and non-toxic character renders it a promising material to be employed in food applications [1515 Jennings JA, Bumgardner JD. Chitosan Based Biomaterials: Fundamentals. Duxford: Woodhead Publishers; 2017.]. Due to its physiological, biological, and pharmacological properties, the use of chitosan in novel areas has grown recently, such as in cosmetics and food industries and also in the production of semi-permeable membranes [1616 Goosen MFA. Applications of Chitin and Chitosan. Boca Raton: CRC Press; 1997.].

Due to the antimicrobial character, chitosan is being widely studied in the production of biodegradable films and active packaging [1717 Elsabee MZ, Abdou ES. Chitosan based edible films and coatings: A review. Mat. Sci. Eng. 2013; 33:1819-41.

18 Niamsa N, Baimark Y. Preparation and Characterization of Highly Flexible Chitosan Films for Use as Food Packaging. Am. J. Food Technol. 2009; 4:162-9.
-1919 Taketa TB. Recobrimentos antimicrobianos e nanoestruturados contendo quitosana produzidos pela técnica de automontagem (Layer-by-Layer) para substratos têxteis [thesis]. Campinas: Universidade Estadual de Campinas; 2017.], which allow the slow release of a preservative or an antimicrobial agent on the surface of the food, inhibiting the growth of microorganisms. The chitosan may also act as a crosslinker, rendering the films more resistant, flexible, and with less wear, extending its durability and, consequently, the product shelf life [2020 Shahidi F, Arachchi JKV, Jeon Y. Food applications of chitin and chitosan. Trends Food Sci. Technol. 1999; 10:37-51.].

Brink and coauthors [2121 Brink I, Šipailienė A, Leskauskaitė D. Antimicrobial properties of chitosan and whey protein films applied on fresh cut turkey pieces. Int. J. Biol. Macromol. 2019; 130:810-7.], working with whey-chitosan films, reported an increase of about six days in the shelf-life of turkey meat packed in the film; the authors also reported an inhibition of the growth of the microorganisms L. sakei, L. plantarum, and C. jejuni, and a reduction of the total bacteria count in relation to the no-packed turkey meat. Shojaei and coauthors [2222 Shojaei M, Eshaghi M, Nateghi, L. Characterization of hydroxypropyl methyl cellulose-whey protein concentrate bionanocomposite films reinforced by chitosan nanoparticles. J. Food Process. Pres. 2019; 43:e14158-e70.], working with composite cellulose-whey films with addition of chitosan nanoparticles (CNP), reported an increase of films elastic modulus, and a decrease of flexibility and water permeability. The addition of CNP also improved the films antimicrobial properties, with more pronounced effect on Gram-positive bacteria.

Shokri and Kamkar [2323 Shokri Z, Kamkar A. Study the antioxidant activities of edible chitosan and whey protein isolate films containing Mentha pulegium essential oil. Carp. J. Food Sci. Technol. 2018; 10:150-5.] evaluated the antioxidant properties of chitosan and whey, in which both whey-based and chitosan-based films presented very similar antioxidant activity, both regarding DPPH radical scavenging, and inhibition of linoleic acid oxidation. The authors also cited briefly the antimicrobial activity of chitosan and whey, without any evaluation.

Ye and Chen [2424 Ye L, Chen H. Characterization of the interactions between chitosan/whey protein at different conditions. Food Sci. Technol. 2019; 39:163-9.], who studied the interactions of chitosan-whey at different pH and heating conditions, reported a substantial influence of pH in both chitosan and whey microstructures, especially regarding film formation. According to the authors, at pH 4.0, the addition of small amounts of chitosan/whey protein isolate (C/WPI) at the ratio of 1:5 prevented the denaturation of the whey proteins after heating; higher amounts (1:2) of C/WPI led to a depletion flocculation. At a pH range of 5.5-6.0, the combination of chitosan and whey proteins was attractive form an electrostatic standpoint. With small chitosan contents, the complexes formed at pH 5.5 presented higher viscosity, complexes formed at pH 6.0 showed a shear-thinning behavior. With higher chitosan contents, the complexes showed high viscosity at pH 6.0. Heating led to a decrease of the viscosity and increase of turbidity of the complexes.

The present work aimed to evaluate the effect of the addition of several chitosan doses on physical-chemical, optical, and biodegradability properties of whey-based biodegradable films.

MATERIAL AND METHODS

Preparation of the films

The whey-based films were prepared from an aqueous solution of 3.0% m/v of whey (whose composition in dry basis was 13.0 wt.% of proteins, 79.1 wt.% of carbohydrates, 0.3 wt.% of lipids, and 4.5 wt.% of ash, obtained from Relat Laticínios Renner S.A., located in the city of Estação, Rio Grande do Sul state, Brazil), 3.0% m/v of commercial gelatin (from animal source, purchased from Dinâmica, Brazil), and 0.6% v/v of glycerol (acting as crosslinker - 85% purity, from Merck), prepared using distilled water.

The solution was homogenized using a magnetic stirrer (ARE, Velp Scientifica) at 50 rpm, for 10 min, at 25±2 °C. The solution was heated in a water bath at 90 °C for 30 min, being stirred at 50 rpm. After, the solution was removed from the bath and allowed to cool to 25±2 °C. A chitosan solution at 1.0 wt.% was prepared by the dissolution of chitosan (Sigma-Aldrich, low-viscosity chitosan, obtained from crab shells, with deacetylation degree of 76%) in a solution of 1.0% v/v acetic acid, according to Ahmad and coauthors [2525 Ahmad AL, Yasin NH, Derek CJC, Lim JK. Optimization of microalgae coagulation process using chitosan. Chem. Eng. J. 2011; 173:879-82.]. After cooling, different volumes of the chitosan solution were added to the whey/gelatin/glycerol solution, to obtain 0.00; 0.15; 1.50; and 3.00 wt.% of chitosan in relation to film weight. The resulting filmogenic solutions were homogenized by stirring at 50 rpm for 30 min; they presented a pH of 4.3 for all formulations. The degassing was carried out using an ultrasound bath (Q335D, Quimis, Brazil) at 40 kHz for 15 min [2626 Isotton FS. Desenvolvimento e caracterização de filmes de amido de milho eterificado com plastificante glicerol, sorbitol e poli (álcool vinílico) [dissertation]. Caxias do Sul: Universidade de Caxias do Sul; 2013.].

The films were prepared by casting, using Petri dishes with diameter of 10 cm, coated with Teflon®. 10 mL of the chitosan-added solution was put in the Petri dishes, and the solution was spread. The spread solutions were air-dried in an air-conditioned room at 20±2 °C and RH of 33±5% for a period of 48 h [2626 Isotton FS. Desenvolvimento e caracterização de filmes de amido de milho eterificado com plastificante glicerol, sorbitol e poli (álcool vinílico) [dissertation]. Caxias do Sul: Universidade de Caxias do Sul; 2013.].

Physical-chemical properties and morphology analysis

The film thickness was measured weekly, up to 22 weeks (five months), using a digital disk micrometer (MDC-SX, Mitutoyo, Japan), by measuring five different points of each sample. The moisture content was determined monthly, during 5 months, according to ASTM E104-02 standard [2727 American Society for Testing and Materials. ASTM E104-02. Standard Practice for Maintaining Constant Relative Humidity by Means of Aqueous Solutions. West Conshohocken: ASTM International; 2012.]. The mechanical properties of the films were measured monthly, during 5 months, using an Emic texturometer, model DL2000, according to ASTM D882-10 standard [2828 American Society for Testing and Materials. ASTM D882-10. Standard Test Method for Tensile Properties of Thin Plastic Sheeting. West Conshohocken: ASTM International; 2010.], with the following adaptions: the film samples measured 70 x 20 mm; the initial separation between claws was 50 mm; and the traction speed was 1 mm/s, with a load cell of 30 N.

The microstructure (film surface and transversal area) characterization of the films was carried out by analyzing the samples surface using an EDS-scanning electron microscope (SSX 550, Shimadzu, Japan), with an acceleration voltage of 5 kV. The samples were prepared by metallization with a small gold layer. The films samples were analyzed with magnifications of 1000x. To obtain clean breaking points to evaluate the film microstructure throughout thickness, the films were frozen with liquid nitrogen and broken manually.

Optic properties

The visual aspect of the films was evaluated by simple visual inspection, being evaluated the surface homogeneity, film uniformity, color changes, and visible growth of fungi. The opacity was measured using a UV-Visible spectrophotometer (DU-530, Beckman Coulter, USA), being measured the absorbance (Abs) at the wavelength of 600 nm. The opacity was calculated by the formula: Opacity=Abs/X (where ‘X’ is the film thickness in millimeters), proposed by Han and Floros [2929 Han JH, Floros JD. Casting antimicrobial packaging films and measuring their physical properties and antimicrobial activity. J. Plast. Film Sheeting. 1997; 13:287-98.] and Ramos and coauthors [3030 Ramos OL, Reinas I, Silva SI, Fernandes JC, Cerqueira MA, Pereira RN, Vicente AA, Poças MF, Pintado ME, Malcata FX. Effect of whey protein purity and glycerol content upon physical properties of edible films manufactured therefrom. Food Hydrocoll. 2013; 30:110-22.]. The films were evaluated weekly, up to 22 weeks (about 5 months).

Shelf life test

The prepared films were exposed to the environment, inside the Laboratory of Materials and Membranes (LAMEM) of the university, which has had an average temperature of 18 °C (max. 21 °C and min. 15 °C) and an average RH of 80% (max. 96% and min. 64%); the films were exposed to sunlight and also to microbial contamination by airborne microorganisms. The films were tested for 150 days (5 months). The opacity, elongation at break, moisture content, thickness, and microscopic morphology of the films were evaluated during this period.

Biologic degradation

For the biologic degradation test, film samples with dimensions of 60 x 40 mm were prepared. A ‘simulated soil’ was prepared, following the ASTM G160-12 standard [3131 American Society for Testing and Materials. ASTM G160-12. Standard Practice for Evaluating Microbial Susceptibility of Nonmetallic Materials by Laboratory Soil Burial. West Conshohocken: ASTM International; 2019.]. Each film sample was put inside a polypropylene screen package with 80 x 100 mm. The simulated soil was put in twelve transparent polypropylene cups with 300 mL capacity; each film was evaluated in three replicates. The packages were put in the simulated soil, allowing contact between the soil and the films. The film samples were incubated for eight days, inside a common greenhouse. The films were weighed every day until the end of the incubation time.

The pH, volatile solids, total C, N, and P contents of the test soil were evaluated before and after the incorporation of the films. The pH was analyzed in accordance with ASTM D4972-19 standard [3232 American Society for Testing and Materials. ASTM D4972-19, Standard Test Methods for pH of Soils. West Conshohocken: ASTM International; 2019.], the volatile solids content was determined following the SMEWW 2540-G standard [3333 Standard Methods for the Examination of Water and Wastewater. Method 2540-G - Solids. Washington DC, 2017.], total P content was analyzed following SMEWW-4500 P, B and E standards [3434 Standard Methods for the Examination of Water and Wastewater. Method 4500 P, B and E. Washington DC, 2017.], and both total C and N contents were determined in accordance with the methods described by Tedesco and coauthors [3535 Tedesco MJ, Gianello C, Bissani CA, Bohnen H, Wolkweiss SJ. Análises de solo, plantas e outros materiais. 2nd ed., Universidade Federal do Rio Grande do Sul: Porto Alegre; 1995.].

Experimental design and statistical analysis

Each film formulation was prepared in four replicates. Each replicate sample was used in both physical-chemical, mechanical and optic characterization of the films. The obtained data underwent analysis of variance (ANOVA) and the means were compared by Tukey’s multiple range test at 5% probability (α = 0.05).

RESULTS AND DISCUSSION

Characterization of the mechanical and optical properties of the films

The whey solution was used because it is a byproduct stream of dairy industry, and, therefore, do not require water removal when compared to whey protein concentrate as a feedstock. The commercial gelatin was added to improve the film plasticity and elasticity. The visual aspect of the whey-based films was related to their appearance during exposure period to the environment. Fresh-prepared films were translucid, without defects or cracks, with a yellowish hue to naked eye. Figure 1 presents the fresh-prepared films.

Figure 1
Visual aspect of the fresh-prepared films. A - film without chitosan; B - addition of 0.15 wt.% chitosan; C - addition 1.50 wt.% chitosan; D - addition 3.00 wt.% chitosan.

Considering film thickness, it is noteworthy the similarity of the behavior among the formulations. Once film thickness is related to the volume of filmogenic solution that is put in each Petry dish for casting, there was no important difference in film thickness after long periods of environmental exposure. Table 1 presents the statistical analysis for the film thickness in relation to the formulations and storage time.

Table 1
Statistical analysis of film thickness in relation to the film formulation and storage time (CV = 5.21%).

Considering film formulation, the one with 3.00 wt.% presented the highest thickness, probably due to moisture retention. However, the formulation of 0.00 (chitosan-free) and 1.50 wt.% have not differed statistically, indicating that the preparation procedure and/or filmogenic solution properties during film casting may influence the final film thickness.

For the storage time, there is no important difference among the measurements; only in the first week after film preparation there was a significant difference from the other weeks. This may be result of film rearrangement and/or maturation in contact with external moisture, after film saturation/equilibration with the environment the thickness remained nearly unchanged, as observed in Table 1.

All films presented a decrease in thickness in the first week of evaluation, it increased in the second week and stabilized after the first month (fourth week). This variation may be attributed to the drying process employed in film production or even a possible moisture absorption by the films. It was observed a more unstable behavior of film thickness in the initial weeks, when the films have the highest water content in their formulation after drying and film formation; this can be seen by analyzing the moisture content of the material and by differences in the film flexibility.

Comparing the thicknesses of the fresh-prepared films, Leceta and coauthors [3636 Leceta I, Arana, P, Guerrero P, De La Caba K. Structure-moisture sorption relation in chitosan thin films. Mater. Lett. 2014; 128:125-7.] reported for chitosan-based films an average thickness of 64.7±3.0 µm. Fráguas and coauthors [3737 Fráguas RM, Simão AA, Faria PV, Queiroz ER, Junior ENO, Abreu CMP. Preparo e caracterização de filmes comestíveis de quitosana. Polímeros. 2015; 25:48-53.] reported, for films containing 1.5 wt.% chitosan, thickness ranging from 60 to 68 µm. Considering the fresh-prepared films produced in the present work presented thickness ranging from 62 to 85 µm, it can be seen that the average thickness of the present films was similar to other works.

Concerning the mechanical properties of the films, there were differences among the formulations. Increasing values of chitosan decreased the elongation at break. The chitosan-free film presented a percentual elongation of 49.01%, whereas the film with 3.0 wt.% chitosan presented a percentual elongation of 28.60%. Table 2 presents the statistical analysis regarding the films’ elongation at break.

Table 2
Statistical analysis of the elongation at break (%) considering film formulation and storage time (CV = 17.41%).

As can be seen in Table 2, for film formulation, only in the months ‘0’ and ‘2’ there was statistical difference between treatments. In month ‘0’ the chitosan-free film presented the highest elongation (49.01%), whereas the film with 3.00 wt.% chitosan had the lowest (28.60%). However, in month ‘2’, the chitosan-free film presented the lowest elongation (5.30%), followed by the film with 3.00 wt.% chitosan (6.86%); the other two formulations (0.15 and 1.50 wt.% chitosan) have had similar elongation values (11.99 and 10.49%, respectively). For the storage time, only in month ‘0’ there was an important difference from the other months; from month ‘1’ on there was no statistical difference in the formulations, with exception of the 0.15 wt.% chitosan, which presented a statistical difference on month ‘2’.

Enhancement of film and membrane flexibility is directly related to the content of plasticizer agents within the polymeric structure, weakening the intermolecular forces in the three-dimensional structure and allowing wider movements among the polymeric chains. Some plasticizers have as a characteristic to decrease the film/membrane elastic modulus and increase the strain percentage due to an increase in water absorption by the biopolymer (moisture content); this may contribute for a wider mobility in a chitosan-added film [3838 Martínez-Camacho AP, Cortez-Rocha MO, Ezquerra-Brauer JM, Graciano-Verdugo AZ, Rodriguez-Félix F, Castillo-Ortega MM, Yépiz-Gómez MS, Plascencia-Jatomea M. Chitosan composite films: Thermal, structural, mechanical and antifungal properties. Carbohydr. Polym. 2010; 82:305-15.-3939 Azevedo EP, Saldanha TDP, Navarro MVM, Medeiros AC, Ginani MF, Raffin FN. Mechanical properties and release studies of chitosan films impregnated with silver sulfadiazine. J. Appl. Polym. Sci. 2006; 102:3462-70.]. According to Azevedo and coauthors [3939 Azevedo EP, Saldanha TDP, Navarro MVM, Medeiros AC, Ginani MF, Raffin FN. Mechanical properties and release studies of chitosan films impregnated with silver sulfadiazine. J. Appl. Polym. Sci. 2006; 102:3462-70.], the use of glycerol as a plasticizer agent induces an increase of film flexibility and elasticity (smaller elastic modulus), this may explain the higher elongation at break of all formulations.

As reported by several previous works, chitosan-based films presented themselves fragile and brittle, therefore, plasticizer agents must be employed to promote better flexibility and malleability. The more brittle behavior (lower elongation at break percentages) for the films with higher chitosan contents may be explained by this phenomenon, where chitosan may be clustering and/or interacting with the polymeric base (whey) in a more crystalline way [4040 Cheung MK, Wan KPY, Yu PH. Miscibility and morphology of chiral semicrystalline poly-(R)-(3-hydroxybutyrate)/chitosane and poly-(R)-(3-hydroxybutyrate-co-hydroxyvalerate)/chitosan blends studied with DSC, 1H T1 and T1F CRAMPS. J. Appl. Polym. Sci. 2002; 86:1253-8.

41 Suyatma NE, Copinet A, Coma V, Tighzert L. Mechanical and barrier properties of biodegradable films made from chitosan and poly(lactic acid) blends. J. Polym. Environ. 2004; 2:1-6.
-4242 Fiori APSM, Gabiraba VP, Praxedes APP, Nunes MRS, Balliano TL, Silva RC, Tonholo J, Ribeiro AS. Preparação e caracterização de nanocompósitos poliméricos baseados em quitosana e argilominerais. Polímeros. 2014; 24:628-35.].

Taking into account that a more brittle property is associated to lower elongation at break, it can be seen that the formulation with 1.5 and 3.0 wt.% of chitosan presented themselves more brittle; in this case the chitosan may act as a barrier to defect flow within film structure, reducing its plasticity and rendering it more brittle [2222 Shojaei M, Eshaghi M, Nateghi, L. Characterization of hydroxypropyl methyl cellulose-whey protein concentrate bionanocomposite films reinforced by chitosan nanoparticles. J. Food Process. Pres. 2019; 43:e14158-e70.]. Smaller stresses at break may also indicate a more fragile film structure, in which the storage time may have deleterious effect on film stabilization, being by moisture reduction or by oxidative processes due to exposure to atmospheric oxygen [4343 Thakhiew W, Devahastin S, Soponronnarit S. Effects of drying methods and plasticizer concentration on some physical and mechanical properties of edible chitosan films. J. Food Eng. 2010; 99:216-24.].

During the environmental exposure test, all films presented a similar behavior, with a steadily reduction rate of elongation at break in the first month. From the first to the third month there was a relatively quadratic trend, with a local maximum at approximately 70 days; this parameter stabilized after approximately three months. Afterward, there was a small trend of increase. After five months (150 days) the elongation percentages ranged between 6.70% (chitosan-free film) and 10.39% (film with 1.5 wt.% chitosan).

The decreasing trend of the elongation at break may be attributed both to a surface crystallization and to moisture removal (water evaporation) of the films during the storage test. Suyatma and coauthors [4444 Suyatma NE, Tighzert L, Copinet A, Coma V. Effects of hydrophilic plasticizers on mechanical, thermal, and surface properties of chitosan films. J. Agric. Food Chem. 2005; 53:3950-7.], who studied the effects of hydrophilic plasticizer agents on thermal, mechanical, and surface properties of chitosan-based films, reported a similar behavior.

Film opacity, which was evaluated weekly, presented absorbance values at the wavelength of 600 nm ranging from 1.380±0.180 Abs600/mm for the films with 0.15 wt.% chitosan to 11.136±0.749 Abs600/mm for the films with 3.00 wt.% chitosan. Table 3 presents the statistical analysis of the opacity for the formulations and the storage time.

Table 3
Statistical analysis for opacity values regarding film formulation and storage time (CV = 6.89%).

By analyzing Table 3, it is possible to verify that, in the first weeks after film preparation, there was no statistical change of the opacity in function of the formulation. However, from about the fourth/fifth week on there was a clear difference among the films. The ones with 1.50 and 3.00 wt.% had similar behaviors, having higher opacity values, with a yellowish hue. For the films with zero and 0.15 wt.% chitosan, the opacity values were lower, but for these two formulations there was no statistical difference between them.

Considering the storage time, a similar trend was observed, in which the opacity values stabilized approximately in the fifth/sixth week, not differing statistically among measurements from week 6 up to the end of the experiment (week 22).

By analyzing Table 3, it is also possible to verify an increase in film opacity of all formulations during the environmental exposure. The opacity values stabilized after approximately six weeks; from this period of time on it is possible to observe higher opacity values for the formulations with 1.50 and 3.00 wt.% chitosan, the formulation with 0.15 wt.% chitosan presented practically the same opacity of the film without chitosan.

According to Chen [4545 Chen H. Functional properties and applications of edible films made of milk protein. J. Dairy Sci. 1995; 78:2563-83.], biopolymer opacity is a result of the material morphology. Fakhoury and coauthors [4646 Fakhoury FM, Martelli SM, Bertan LC, Yamashita F, Mei LHI, Queiroz FPC. Edible films made from blends of manioc starch and gelatin - Influence of different types of plasticizer and different levels of macromolecules on their properties. LWT - Food Sci. Technol. 2012; 49:149-54.] cited that amorphous materials enhance light transmittance, presenting less opacity and, consequently, being more transparent when compared to materials with a more crystalline structure in the morphology. The influence of chitosan addition on films was also cited in other works, such as the ones from Pelissari and coauthors [4747 Pelissari FM, Yamashita F, Garcia MA, Martino MN, Zaritzky NE, Grossmann MVE. Constrained mixture design applied to the development of cassava starch-chitosan blown films. J. Food Eng. 2012; 108:262-7.], Bourtoom and Chinnan [4848 Bourtoom T, Chinnan MS. Preparation and properties of rice starch-chitosan blend biodegradable film. Food Sci. Technol. 2008; 41:1633-41.], Chillo and coauthors [4949 Chillo S, Flores S, Mastromatteo M, Conte A, Gerschenson L, Del Nobile MA. Influence of glycerol and chitosan on tapioca starch-based edible film properties. J. Food Eng. 2008; 88:159-68.], and García and coauthors [5050 García MA, Pinotti A, Martino NM, Zaritzky NE. Characterization of composite hydrocolloid film. Carbohydr. Polym. 2004; 56:339-45.], who employed chitosan as an additive to other polymers, such as cassava, tapioca, and rice starch and methylcellulose.

Relative to the moisture content of the films, there was a trend of decrease with storage. The fresh-prepared films presented moisture contents that ranged between 17.9 wt.% (chitosan-free film) and 19.7 wt.% (film with 3.00 wt.% chitosan). The statistical analysis of the moisture content is presented in Table 4.

Table 4
Statistical analysis of moisture content considering film formulation and storage time (CV = 9.38%).

According to Table 4, the average moisture content has not differed among all formulations; only the formulations of 0.15 and 3.00 wt.% presented statistical difference. Relative to the storage time, it was clear a decreasing trend, in which the first two months presented the highest moisture contents, with reduction of the film moisture, probably due to water loss to the environment. Despite some variation between the months that may be result of variation of the environmental humidity, the moisture content of the films stabilized from the second month on.

According to Rindlay-Westling and coauthors [5151 Rindlay-Westling A, Stading M, Hermansson A, Gatenholm P. Structure, mechanical and barrier properties of amylose and amylopectin films. Carbohydr. Polym. 1998; 36:217-24.] and Leceta coauthors [3636 Leceta I, Arana, P, Guerrero P, De La Caba K. Structure-moisture sorption relation in chitosan thin films. Mater. Lett. 2014; 128:125-7.], the presence of glycerol (plasticizer agent) favors water absorption due to its intrinsic hygroscopicity. The relatively low differences among formulation may also be result of the storage conditions, which were an average temperature of 18 °C and relative humidity of 80%.

During storage, it was observed an important reduction in the moisture content of the films. This reduction may be a result of water diffusion/releasing to the environment as a slower and progressive drying process, with the removal of the remaining solvent trapped inside the polymeric structure. When reaching an equilibrium, the moisture content tends to stabilize; this was observed after approximately 70 days of storage. This change in the moisture content also affects the mechanical properties, as was observed with the elongation at break of the films.

Films surface microstructure characterization

The films surface microstructure was evaluated by SEM. Relative to the fresh-prepared films, it could be observed that the chitosan-free film and the film with 0.15 wt.% chitosan presented a relatively smooth surface, with the presence of small pores and other minor defects, very probably being result of crystallization of the polymeric compounds during film drying or even a poor homogenization of the filmogenic material. Figure 2 presents the SEM images of the films surface and transversal section (thickness) for all formulations.

Figure 2
SEM analysis (1000x magnification) of the films surface and transversal area (thickness). A - film without chitosan; B - addition of 0.15 wt.% chitosan; C - addition 1.50 wt.% chitosan; D - addition 3.00 wt.% chitosan.

In the films with higher chitosan contents, it was possible to observe surfaces with less homogeneity, more and larger pores, and more defects. This may be linked to the intrinsic crystallinity of chitosan, which may induce more pore and defect formation. Considering the chitosan used in the present work had a low purity, the presence of other substances may act as nucleation centers that induce pore and defect formation in both film surface and through the internal structure. Alberti and coauthors [5252 Alberti ML, Souza SJ, Gonçalves H, Yamashita F, Shirai MA. Effect of chitosan addition in starch and poly(lactic acid) sheets produced by extrusion. Braz. J. Food Res. 2015; 6:80-90.], working with addition of chitosan in starch and polylactic acid-based films, also reported the presence of clusters similar than the ones that occurred in the present work.

Considering the film with the lower chitosan content (0.15 wt.%), it presented itself more homogeneous (less pores and defects) when compared to the films with higher chitosan content. During storage was possible to see the presence of crystals that may be formed due to arrangements in the film structure as a result of moisture reduction (Figure 2).

Taking into account that chitosan has a semicrystalline profile, which is result of the strong intermolecular and intramolecular interactions, mainly originated from the hydrogen bonds among the hydroxyl, amino, and amide functional groups presented in the structure of chitosan. This favors a more compact and ordered organization of the polymeric chain, with some degree of crystallinity [5353 Oliveira RA. Síntese e caracterização de compósito quitosana com carbonato de cálcio extraído de casca de ovo para possíveis aplicações como curativo. Proceedings of the 1st Encontro Nacional de Educação. Campina Grande: Universidade Federal de Campina Grande; 2011.].

It can be assumed that a possible presence of clusters in the film without chitosan may be related to a poor homogenization of the filmogenic solution during film preparation, or an incomplete denaturation and dispersion of the whey proteins. According to Le Tien and coauthors [5454 Le Tien C, Letendre M, Ispas-Szabo P, Mateescu MA, Delmas-Patterson G, Yu HL, Lacroix M. Development of biodegradable films from whey proteins by cross-linking and entrapment in cellulose. J. Agric. Food Chem. 2000; 48:5566-75.], whey proteins have the capacity to crystallize during heating in film preparation. Abdelwahab and coauthors [5555 Abdelwahab MA, Flynn A, Chiou B, Imam S, Orts W, Chiellini E. Thermal, mechanical and morphological characterization of plasticized PLA-PHB blends. Polym. Degrad. Stabil. 2012; 97:1822-8.] and Souza and coauthors [5656 Souza G, Santos A, Vinhas G. Avaliação das propriedades da blenda de poli(3-hidroxibutirato) /quitosana após esterilização térmica ou radiolítica. Polímeros. 2016; 26:352-9.] also reported the influence of chitosan on the morphological characteristics of films based on biopolymers, with similar results.

Shelf life test

During the shelf life test, the films presented an increasing yellowish color; the ones with higher chitosan contents (1.50 and 3.00 wt.%) showed themselves more colored than the others. This also corresponded with the increasing opacity of the films, observed in the present work and also cited by Silva and coauthors [5757 Silva MG, Silva SB, Silva J, Santin CK, Souza D, Frota Jr. M. Caracterização de filmes à base de quitosana. Proceedings of the 9th Congresso Brasileiro de Engenharia Química em Iniciação Científica. São Paulo: Universidade de São Paulo; 2015, p. 1710-5.], who reported more intense yellowish color with increase of the chitosan dose. The films presented themselves intact, without cracks, but developed a bent shape during environmental exposure. Figure 3 presents the films soon after production and after 30, 75, and 150 days of environmental exposure.

Figure 3
Visual aspect of the films during shelf life test. A - film without chitosan; B - addition of 0.15 wt.% chitosan; C - addition 1.50 wt.% chitosan; D - addition 3.00 wt.% chitosan.

However, with increase of the testing time, the films presented a more brittle behavior. This may be result of moisture reduction or even result of exposure to atmospheric oxygen, that may cause oxidative stress in the film structure. Considering the behavior of the films during storage, although there was a worsening of some properties (elongation at break, increased brittleness), there was an overall stabilization of the films’ properties, especially moisture content and thickness.

Biological degradation of the films

A test soil was prepared in accordance to ASTM standard G160-12 [3131 American Society for Testing and Materials. ASTM G160-12. Standard Practice for Evaluating Microbial Susceptibility of Nonmetallic Materials by Laboratory Soil Burial. West Conshohocken: ASTM International; 2019.] to be employed in the degradation tests of the films and to evaluate the physical-chemical changes in the reference soil due to film degradation and incorporation. Table 5 presents some physical-chemical parameters of the test soil prior and after film incorporation.

Table 5
Change of test soil physical-chemical parameters with film degradation and incorporation.

According to ASTM G160-12 standard [3131 American Society for Testing and Materials. ASTM G160-12. Standard Practice for Evaluating Microbial Susceptibility of Nonmetallic Materials by Laboratory Soil Burial. West Conshohocken: ASTM International; 2019.], the pH range of the soil must fall between 6.5 and 7.5, to provide microorganism survival; in the present work, the soil pH ranged from 6.88, prior to film incorporation, to 6.99 to the film-incorporated soil, keeping within the recommended pH range.

Beyond pH, carbon and nitrogen contents are also important for microbial growth; shortage of any of these elements may hinder the microbial activity, delaying the degradation of any disposed material. According to Pereira and Fialho [5858 Pereira LAA, Fialho ML. Gestão da sustentabilidade: compostagem otimizada em resíduos sólidos orgânicos com a utilização de metodologia enzimática na implantação de uma usina de compostagem de lixo no município de santa juliana/MG. Int. J. Knowl. Eng. Manag. 2013; 2:52-85.], a C/N ratio of 30 is considered optimal for microbial growth. In the present work the starting C/N relation of the test soil was 18.06; the soil C/N relation at the end of the experiment was 24.59, indicating an increase in this ratio due to carbon incorporation from the film.

Due to film composition, the increase in C/N ratio is a normal result of the film incorporation by the soil. It is noteworthy that there was no change in the total N content (from 0.35 to 0.34 wt.%), this may be result of N biotransformation from the organic to ammoniacal and nitrate forms, which are readily absorbed by plants and the soil microbiota, or even a biotransformation to dinitrogen (N2), leaving the soil to the atmosphere. It is also possible to verify a near steady-state behavior of N in the soil, characteristic of a relatively stabilized soil [5959 Cantarella H. Nitrogênio. In: Novais RF, editor. Fertilidade do solo. Viçosa: Sociedade Brasileira de Ciência do Solo; 2007. p. 375-470.]. Relative to P soil content, it presented a slight decrease (from 0.24 to 0.21 wt.%) with film incorporation. This may be result of microbial activity (nutrient consumption) or due to the increase in soil pH, which reduces P availability [3535 Tedesco MJ, Gianello C, Bissani CA, Bohnen H, Wolkweiss SJ. Análises de solo, plantas e outros materiais. 2nd ed., Universidade Federal do Rio Grande do Sul: Porto Alegre; 1995.,6060 Sato S, Comerford NB. Influence of soil pH on inorganic phosphorus sorption and desorption in a humid Brazilian Ultisol. Rev. Bras. Cien Solo. 2005; 29:685-94.].

The volatile solids content is a way of measuring the organic matter (OM) content, which includes the products of biological degradation of the whey-based films. It was observed an increase of the volatile solids content from 17.18 wt.% in the soil prior to film incorporation to 18.63 wt.% in the film-incorporated soil. Considering the increase in the volatile solids of the soil indicate that the microbial degradation of the films was quite quick.

Visual characterization

The visual aspect and integrity of the films were evaluated daily during the exposure time to the soil. The visual aspect of the four formulated films for the eight days of testing is presented in the Figure 4.

Figure 4
Visual aspect and degradation behavior of the films during exposure to the test soil. A - film without chitosan; B - addition of 0.15 wt.% chitosan; C - addition 1.50 wt.% chitosan; D - addition 3.00 wt.% chitosan.

It can be seen that all films, with and without chitosan, presented similar behavior in the soil exposure test. The color change in the second day is a strong indicative of the degradability of the films, for all formulations. From day five on, it is visible the disaggregation of the structure of the films, they starting to tear apart. The addition of chitosan presented no visible inhibition of the microbial activity on the films, being all formulation very degradable when exposed to soil (considering the test soil), being nearly completely decomposed after eight days.

It was also observed a strong aggregation of soil particles to the films, must probably due to the high hydrophilicity and flexibility of the films. According to Shah and coauthors [6161 Shah AA, Hasan F, Hameed A, Ahmed S. Biological degradation of plastics: A comprehensive review. Biotechnol. Adv. 2008; 26:246-65.], visual changes in the surface of the films, such as pores, cracks, disaggregation, or color change does not prove definitely the occurrence of a microbial/biological degradation from a metabolic point of view; however, they can be assumed as the first signs of microbial activity on the films.

Considering that all films lost started to disaggregate in the fifth day, the films with higher chitosan contents aggregated more amounts of soil in their surfaces. According to Tonhi and Peplis [6262 Tonhi E, Peplis AMG. Obtenção e caracterização de blendas colágeno-quitosana. Quím. Nova. 2002; 25:943-8.] and Shah and coauthors [6161 Shah AA, Hasan F, Hameed A, Ahmed S. Biological degradation of plastics: A comprehensive review. Biotechnol. Adv. 2008; 26:246-65.], this is result of chitosan high hydrophilicity, result of the presence of hydroxyl and amino groups in the polysaccharide structure. Gigli and coauthors [6363 Gigli M, Negroni A, Soccio M, Zanaroli G, Lotti, N, Fava F, Munari A. Influence of chemical and architectural modifications on the enzymatic hydrolysis of poly(butylene succinate). Green Chem. 2012; 14:2885.] and Paoli [6464 Paoli MA. Degradação e estabilização de polímeros. São Paulo: Chemkeys; 2008.] cited a possible correlation between the biological degradation rate of a biodegradable film and its hydrophilicity degree.

Li and Chen [6565 Li J, Chen H. Biodegradation of Whey Protein-Based Edible Films. J. Polym. Environ. 2000; 8:135-43.], investigating the biological degradation of concentrated whey-based (CW) and isolated whey-based (IW) films, concluded that, when in contact with soil, the CW films were degraded more quickly than the IW ones. The IW films started to decompose after two days of incubation in the soil; after seven days, the mass loss of the films was more than 80%, indicating a nearly complete decomposition of the films.

Evaluation of loss mass of the films

The mass of the soil-exposed films was measured daily to verify the behavior of the mass loss of the films during biological decomposition. Table 6 presents the statistical analysis of the mass loss of the films regarding formulation and day of exposure.

Table 6
Statistical analysis of the mass loss of the films regarding film composition and days of exposure (CV = 8.79%).

Regarding film composition, it can be seen that for the films with 1.50 and 3.00 wt.% chitosan the total mass presented itself slightly higher than in other formulations; this may be result of water (moisture) capture due to chitosan high hydrophilicity. In relation to the days of exposure, there was an increase in film mass up to day two; this may be result of moisture absorption of even due to the strong aggregation of soil particles to the film. The decrease due to degradation occurred steadily for all formulations, but without important statistical difference in the last days. According to Brambilla [6666 Brambilla VC. Avaliação das propriedades mecânicas, térmicas, morfológicas, estruturais e a degradação de compósitos de Poli(Ácido Lático)/Buriti [dissertation]. Caxias do Sul: Universidade de Caxias do Sul; 2013.], a mass increase is the first step towards biological degradation of a material, being a result of water absorption and, afterward, the starting of microbial colonization of the material surface and formation of the biofilm.

Tonhi and Peplis [6262 Tonhi E, Peplis AMG. Obtenção e caracterização de blendas colágeno-quitosana. Quím. Nova. 2002; 25:943-8.] cited the proportional relation between the chitosan amount added to a film and the capacity it to aggregate soil particles; this can be seen especially by analyzing Table 6, in which the formulation with 3.00 wt.% chitosan presented higher mass values when compared to other formulations. Assis and Silva [6767 Assis OBG, Silva VL. Caracterização estrutural e da capacidade de absorção de água em filmes finos de quitosana processados em diversas concentrações. Polímeros. 2003; 13:223-8.] reported that increasing amounts of chitosan added to biodegradable films interfere significantly in the water amounts absorbed. The hydrophilicity of chitosan is considered the main reason for this phenomenon, considering that the hydroxyl and amino groups present strong affinity for polar molecules, such as water. This helps water absorption and diffusion throughout the film structure [3636 Leceta I, Arana, P, Guerrero P, De La Caba K. Structure-moisture sorption relation in chitosan thin films. Mater. Lett. 2014; 128:125-7.].

The predominance of amino groups in chitosan, which present covalent N-H bonds, generate sites of high polarity, which are places for alignment and rearrangement of water molecules. This characteristic is also associated to acetamido groups, which also present polar character, and confer chitosan a biopolymer with a high degree of hydrophilicity [6767 Assis OBG, Silva VL. Caracterização estrutural e da capacidade de absorção de água em filmes finos de quitosana processados em diversas concentrações. Polímeros. 2003; 13:223-8.].

When the soil amount aggregated by the film reached its capacity, it was observed a reduction of film mass for all formulations, especially from day three on. The mass reduction, which indicated the biotransformation of the polymeric structure of the film, eventually led to the disaggregation of the entire structure, the films crumbling due to the weakened and decomposing polymeric chains; the presence of moisture in the soil may also help decomposition by enhancing the decomposition rate and also by helping solubilizing the smaller fragments of the films [6868 Jooyandeh H. Whey Protein Films and Coatings: A Review. Pak. J. Nutr. 2011; 10:296-301.].

After eight days of analysis, despite the mass of the decomposed film was similar to the starting mass, the presence of fragments of aggregated soil may induce a false idea of no (or little) mass loss. By analyzing Figure 4, it can be seen that the films were nearly completely decomposed. This indicated that whey-based films with addition of chitosan present a very high biodegradability, being nearly completely decomposed after eight days of exposure to an appropriate decomposition medium (soil); however, as seen in the shelf-life test, the films did not decompose even after 150 days (5 months) without and appropriate medium.

CONCLUSION

There were changes in the physical-chemical and mechanical properties of the films, in which increasing chitosan content rendered a lower elongation at break, higher moisture content, opacity and film thickness. Most of the properties remained stable during the storage time (5 months). The addition of chitosan rendered a yellowish hue to the films along with an increase in film opacity, especially the formulations with higher chitosan content. The films with higher chitosan content (1.50 and 3.00 wt.%) presented more pores and surfaces with lower homogeneity; the one with 0.15 wt.% presented the best results considering homogeneity and crystallinity. Considering biological degradation, all films presented a high biodegradability, the ones with higher chitosan content presented a higher soil aggregation due to the increased hydrophilicity. The film degradation increased some soil parameters, such as volatile solids and total organic C; however, it has not changed soil pH, N, and P contents. All films presented a similar behavior concerning microbial degradation, where chitosan addition have not enhanced the film resistance to fungi attack. Taking into account that chitosan is a relatively expensive material, the chitosan content of 0.15 wt.% may be considered the best one to be employed in whey-based biodegradable films.

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HIGHLIGHTS

  • 1
    Dairy production generates large amounts of whey.
  • 2
    Chitosan was added to verify the increase in film resistance to microbial decomposition.
  • 3
    The films stored at room conditions did not decompose even after 5 months.
  • 4
    Soil-exposed films were decomposed in a period of approximately eight days.

  • Funding:

    This research received no external funding.

Publication Dates

  • Publication in this collection
    31 Aug 2020
  • Date of issue
    2020

History

  • Received
    28 Mar 2020
  • Accepted
    10 June 2020
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